Project Summary The current project fulfills the spirit of the R21 application by proposing to examine a largely overlooked aspect of autoimmunity: lymphangiogenesis. In the particular case of type 1 diabetes (T1D), the destructive lesion that affects the islets of Langerhans and leads to the killing of the insulin producing ? cells, is called insulitis. It proceeds in two successive steps, peri-insulitis and infiltrating insulitis, and is made of immunocytes. This influx of immune cells requires lymphatics. Because the islet is normally very poorly vascularized by lymphatics, the appearance of neo lymphatics is required for the progression of disease. We have examined in very young NOD mice the appearance of lymphatic vessels near islets and their progression into organized functional communication with the pancreatic lymph nodes (PLN). In addition, we have also studied the communication between duodenum and PLN and determined that lipophilic substances could readily access PLNs from the intestine. Our application is intended to explore the cellular and molecular aspects of the two facets of the pancreatic lymphatic trafficking for which we have accumulated surprising preliminary data, and to integrate both into a frame that could explain disease onset and progression. To achieve our goal, we have design two separate specific aims that complement each other. Specific aim 1: Physiology of the pancreatic lymph node. We have shown that lipids could be transported directly from duodenum to PLNs through the lymphatic system. We will examine whether this observation expands to nucleic acids and proteins. We will then examine if this direct communication is operational to deliver microbial and/or dietary compounds to the PLN and prime resident autoreactive T cells? To answer these questions, we will combine classic physiology experiments with a sophisticated single cell analysis approach. Our hypothesis is that this unique lymphatic communication is responsible for a cytokine driven activation state that we have observed for PLN CD4 T cells. Its relevance to autoimmunity must be investigated. Specific aim 2: Neo-lymphangiogenesis and T cell migration to pancreatic lymph nodes. We have observed the development of neo lymphatic vessels near islets of young pre-diabetic NOD mice. The initial steps of this phenomenon precede insulitis. What cells and factors are necessary for lymphatic proliferation? Each of the following critical components of lymphatic sprouting can be detected by immunocytochemistry near diseased islets: LYVE-1, podoplanin, VEGF-R3, and Midkine. We hypothesize that two pro-inflammatory cells, the macrophage and the platelet, are central to lymphangiogenesis in the context of T1D. We will test this hypothesis by a series of in vivo experiments combined with state of the art single cell RNAseq techniques. Our studies should place lymphangiogenesis chronologically in the pathology of T1D and help evaluate the possible therapeutic potential of some of the important pro-lymphangiogenic molecules.